Clinical Chemistry
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Clinical Chemistry 54: 1317-1324, 2008. First published June 12, 2008; 10.1373/clinchem.2007.100644
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Right arrow Lipids, Lipoproteins, and Cardiovascular Risk Factors
(Clinical Chemistry. 2008;54:1317-1324.)
© 2008 American Association for Clinical Chemistry, Inc.

Lipids, Lipoproteins, and Cardiovascular Risk Factors

Potential of Sterol Analysis by Liquid Chromatography–Tandem Mass Spectrometry for the Prenatal Diagnosis of Smith-Lemli-Opitz Syndrome

William J. Griffiths1,a, Yuqin Wang1, Kersti Karu2, Emmanuel Samuel2, Shane McDonnell3, Martin Hornshaw3 and Cedric Shackleton4

1 Institute of Mass Spectrometry, School of Medicine, Swansea University, Swansea, UK; 2 The School of Pharmacy, University of London, London, UK; 3 Thermo Fisher Scientific, Hemel Hempstead, UK; 4 Children’s Hospital Oakland Research Institute, Oakland, CA.

aAddress correspondence to this author at: Institute of Mass Spectrometry, School of Medicine, Grove Building, Swansea University, Singleton Park, Swansea SA2 8PP, UK. Fax +44 (0)1792 295554; e-mail w.j.griffiths{at}swansea.ac.uk

Background: Smith-Lemli-Opitz syndrome (SLOS), a severe disorder of cholesterol synthesis, is classically diagnosed prenatally by GC-MS analysis of sterols in amniotic fluid. Considering the current trend toward tandem mass spectrometry (MS/MS) methodologies, we developed prototype LC-MS/MS methods for accurate diagnosis of the disorder.

Methods: 3β-Hydroxysterols in amniotic fluid are oxidized with cholesterol oxidase to their corresponding 3-ketones, which are then derivatized with Girard P (GP) hydrazine in a "one-pot" reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)–MS/MS owing to the presence of a charged quaternary nitrogen and are analyzed by reversed-phase LC-ES-MS/MS. Both capillary and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper-absorbed blood spots.

Results: In a double-blind analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the ratio of 7 + 8-dehydrocholesterol (7 + 8-DHC) to cholesterol was <0.02 [range 0.00–0.02, mean (SD) 0.01 (0.007)] in all control samples (intraassay variation 5.91%) and >0.20 [0.20–1.13, 0.79 (0.35)] in SLOS (intraassay variation 4.56%), corresponding to a difference in ratios between the 2 groups of at least a factor of 10. The limit of quantification was equivalent to that of 2 nL amniotic fluid injected on-column.

Conclusions: We describe a proof-of-concept for the prenatal diagnosis of SLOS. Further developments will be necessary to automate sample handling and reduce chromatographic time for the methodology to be used in pre- and postnatal diagnosis.






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